There is increasing utilization of flow cytometry in both basic and applied research to accomplish diagnostic assays. One such area of research is the investigation of circulating cells that have detached from tumors. Another very active and contemporary area is the generation of flow cytometry side populations that contain multipotent cancer stem cells that can be present in less than 1% of the cell population. Furthermore, it is well known that the flow cytometry characterization of nearly all types of stem cells, including human embryonic stem cells, is a challenge due to the high cell loss during the sorting process. This has led some to consider the inclusion of ROCK inhibitors in the cell sorting protocol to increase viability and improve the detection of stem cell surface markers. To address these challenges CPSI Biotech proposes to develop the first FACS compatible sorting reagent, referred to as FACSGuard, expressly designed to protect cells during the flow cytometry process. This media will be designed to be used at normothermic (37oC) temperatures to ameliorate several stress pathways that are activated during a typical cell dissociation, separation and flow procedure. If successful, this reagent can be used in a variety of flow cytometry applications from low stress Guava to the high PSI (70) often required in more traditional FACS systems to separate other cell types. The Specific Aims of the project are the following: (1) Test a variety of candidate cell stress pathway inhibitors in conditions that mimic those experienced by cells under flow cytometry conditions for possible inclusion in FACSGuard; (2) Develop several versions of FACSGuard; and (3) Test the versions of FACSGuard on two human embryonic stem cell lines as well as other more robust cell types. Phase 2 will be dedicated to using RT-PCR and other microarray techniques to determine if one generic FACSGuard reagent is sufficient for most flow cytometry applications or if several cell and or condition-specific variants should be developed. The successful completion of this project will lead to an improved method of identifying and understanding cancer stem cells found in FACS side populations as well as directly and indirectly improve stem cell therapy applications. PUBLIC HEALTH RELEVANCE: This project is designed to develop a protective solution for stem cells so that both their viability and function can be protected during their separation and analysis. This will have direct benefit to diverse areas ranging from understanding cancer stem cells to stem cell therapy.